Development of active edible coatings based on fish gelatin enriched with Moringa oleifera extract: Application in fish (Mustelus mustelus) fillet preservation

Abstract An edible coating was developed using gelatin extracted from the skin of gray triggerfish (Balistes capriscus) and applied to the fillet of the smooth‐hound shark (Mustelus mustelus). Moringa oleifera leaf extract was added to gelatin coating solution to improve its preservative properties. The phenolic profiles and antioxidant and antibacterial activities of M. oleifera extracts were determined. Phenolic acids constituted the largest group representing more than 77% of the total compounds identified in the ethanol/water (MOE/W) extract, among which the quinic acid was found to be the major one (31.48 mg/g extract). The MOE/W extract presented the highest DPPH• scavenging activity (IC50 = 0.53 ± 0.02 mg/ml) and reducing (Fe3+) power (EC0.5 = 0.57 ± 0.02 mg/ml), as well as interesting inhibition zones (20–35 mm) for the most tested strains. Coating by 3% of gelatin solution significantly reduced most deterioration indices during chilled storage, such as malondialdehyde (MDA), total volatile basic nitrogen (TVB‐N), weight loss, pH, and mesophilic, psychrophilic, lactic, and H2S‐producing bacterial counts. Interestingly, coating with gelatin solution containing MOE/W extract at 20 μg/ml was more effective than gelatin applied alone. Compared with the uncoated sample, gelatin‐MOE/W coating reduced the weight loss and MDA content by 26% and 70% after 6 days of storage, respectively. Texture analysis showed that the strength of uncoated fillet increased by 46%, while the strength of fillet coated with gelatin‐MOE/W only increased by 12% after 6 days of storage. Fish fillet coated with gelatin‐MOE/W had the highest sensory scores in terms of odor, color, and overall acceptability throughout the study period.


| INTRODUC TI ON
Currently there is growing interest in using ecological food packaging based from natural polymers as an alternative to conventional nonbiodegradable polymers. Moreover, the development of "active packaging" by incorporating bioactive compounds with antimicrobial and antioxidant activities into the polymer matrices continues to develop progressively until today. This active packaging can be interesting to preserve perishable food products and thus improve their shelf-life and sensory quality (Araghi et al., 2015;Li et al., 2019;Abdelhedi et al., 2019;Eghbalian et al., 2021;Shahbazi et al., 2021).
Fish gelatin, produced by partial hydrolysis of collagen, is an interesting alternative to mammalian-derived gelatin for the production of edible packaging films. In fact, it can be obtained economically from fish by-products, without consumer concern, as in the case of porcine and bovine gelatin. Fish gelatin, which was tasteless and colorless, has excellent film-forming, biocompatibility, and mechanical properties especially in the presence of certain agents, such as cross-linking adhesion promoters, other proteins and polysaccharides, among others, that improve its functional properties (Alfaro et al., 2014). Besides, gelatin formed an excellent matrix for hosting bioactive compounds, such as plant extract rich in phenolic compounds or essential oils that can be used to develop antioxidant and antimicrobial packaging films (Hanani et al., 2019;Naeeji et al., 2020;Shahbazi et al., 2021;Staroszczyk et al., 2020). In this context, edible gelatin-based films or coatings have been used to preserve foods, such as trout fillets (Eghbalian et al., 2021), cheese (Salem et al., 2021), shrimp (Mirzapour-Kouhdasht & Moosavi-Nasab, 2020), bread (Oliveira et al., 2020), and beef meat (Jridi et al., 2018), in order to improve their quality during storage.
Moringa oleifera Lam. (Moringaceae) that is called the tree of life was originally from India and is widely cultivated in many parts of the world, such as Tunisia, since it adapts to arid and heat-resistant regions. It has been considered as one of the most useful perennial trees due to its high content of nutrients and bioactive compounds without any reported undesirable side effects. The leaves have been used to treat malnutrition and have also been used as fortificant in many food products (Oyeyinka & Oyeyinka, 2018). Besides, several pharmacological properties of M. oleifera have been reported, such as hepatoprotective, neuroprotective, antidiabetic, antiinflammatory, anticancer, and antiviral activities (Gopalakrishnan et al., 2016;Razzaq et al., 2020). However, few studies have been reported regarding the use of this plant into biopolymer matrices.
The use of edible coatings based on gelatin and natural bioactive compounds would be an interesting strategy for improving the quality of food products. Thus, M. oleifera was chosen as a potential plant of bioactive substances. The phenolics profiles and antioxidant and antibacterial activities of different solvent extracts from M. oleifera leaves were determined. Gelatin was extracted from the skin of gray triggerfish (Balistes capriscus) and then was used to develop gelatin-based coatings enriched with M. oleifera extracts. The effect of active gelatin coating on the physicochemical and microbiological quality of smooth-hound shark (Mustelus mustelus) fillets during refrigerated storage was studied.

| Plant material
Moringa oleifera Lam. (Moringaceae) leaves were collected from the oasis of Chenini-Gabes (southeastern Tunisia, characterized by an arid climate) on April 2020. The leaves were air-dried in the shadow, until constancy of the mass (20 days), then ground into fine powder, and stored at ambient temperature in a dry place and in the dark until use.

| Phytochemical analysis and antioxidant activities
The M. oleifera leaves were extracted using three solvents: (i) ethanol 100%, (ii) ethanol/water (50/50, v/v), and (iii) distilled water. Leaves powder (5 g) was macerated in 100 ml of each solvent in a closed Erlenmeyer flask and stirred at 250 rpm for 12 h. Then, the macerate was filtered through Whatman No. 1 filter paper. The same procedure was repeated twice with the obtained residue, and then the extract was lyophilized and kept in the dark at +4°C until further analysis. Finally, three extracts were obtained: (i) ethanol extract (MOE); (ii) ethanol/water (50/50, v/v) extract (MOE/W); (iii) water extract (MOW).
After that, the total phenolics and flavonoids in M. oleifera extracts were measured as described previously (Dewanto et al., 2002).
The total phenolics content was expressed as mg gallic acid equivalent (GAE)/g extract. The flavonoids content was expressed as mg catechin equivalent (CE)/g extract. Moringa oleifera extracts were also analyzed using liquid chromatography-electrospray ionizationtandem mass spectrometry (LC-ESI-MS) as described previously by Jdir et al. (2017). An LC-MS-2020 quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization source and operated in negative ionization mode was used. The identification of phenolics was done by comparing the retention times and the mass spectra with those of authentic standards of highest purity (≥99.0%), which were from Sigma Chemical Co. (St. Louis, MO, USA).
The reducing (Fe 3+ ) power and DPPH• radical-scavenging activity of M. oleifera extracts were measured as described previously (Yıldırım et al., 2001;Zouari et al., 2011) Hinton broth (MHB) and three wells of 0.5 cm deep were made by using a sterile tip. Then, 60 μl of each extract (1 mg/ml) was added to respective wells. Gentamycin was used as positive reference and the negative control was done with sterile water. Prior to incubation, all plates were stored in the dark at 4°C for 2 h to allow diffusion of the extract to the medium without bacterial growth. At the end of incubation period (24 h at 37°C), the antimicrobial activity was determined by measuring the zone of inhibition around the holes in diameter (mm) after incubation. All tests were carried out for three sample replications and the results were averaged.

| Gelatin extraction and coating preparation
The by-product of gray triggerfish (B. capriscus) was obtained after processing the fish from the Sfax market (Sfax, Tunisia). Gelatin was extracted from fish skin as described previously by Jellouli et al. (2011). The skin was washed with tap water and cut into small pieces (1 × 1 cm) and then soaked in 0.05 M NaOH solution at a ratio of 1:5 (m/v). The mixture was stirred for 2 h and the alkaline solution was changed every 30 min. Then, the alkaline-treated skin was washed with distilled water until a neutral pH was obtained, and then subjected to acid treatment, at a ratio of 1:5 (m/v), using few drops of acetic acid to reach a pH of 3.0 over 18 h with gentle stirring. After that, the pH was neutralized using few drops of 6 M NaOH solution and the mixture was incubated at 50°C with continuous stirring for 24 h. Finally, the mixture was centrifuged at 6000g for 20 min to remove insoluble matter and gelatin-containing supernatant was freeze-dried using a freeze-dryer Christ Alpha 1-2 (Bioblock Scientific, Illkirch, France) and stored at 4°C until use. The gelatin coating solution was prepared by mixing 3 g of dried gelatin in 100 ml of distilled water at 40°C for 30 min. In order to prepare the active gelatin solution, the MOE/W extract was dissolved in the gelatin solution at a final concentration of 20 μg/ml.

| Preparation of fish fillet samples
Fresh smooth-hound shark (Mu. mustelus) fillets were purchased from a local fish market (Sfax, Tunisia). Fish fillets were cut into 2 × 2 × 2 cm cubes and divided into three groups according to the

| Characterization of coated fish fillets
2.6.1 | pH measurement Two grams of fish fillet sample were homogenized in 20 ml of distilled water for 2 min. Then, the pH was measured using a pH meter (Hanna Instruments, Póvoa de Varzim, Portugal). The pH of fish samples from each treatment was measured after 1, 2, 4, and 6 days of storage.

| Weight loss
The weight loss of fish fillet was calculated using the Equation (1).
where W 0 is the initial weight of fish sample and W i is the weight of the same sample after 1, 2, 4, and 6 days of storage.

| Color measurement
Color was measured using a colorimeter (Konica Minolta, Osaka, Japan) with D65 illuminant. The instrument was standardized using a standard white plate. Color was measured for fish samples from each where L*, a*, and b* are the color parameters of the fish samples; L * c , a * c , and b * c are the color parameters of the uncoated fish fillet samples on the first day of storage.

| Texture profile analysis (TPA)
The TPA parameters (strength, cohesiveness, springiness, and chewiness) were measured according to the method described previously by Jridi et al. (2015) using a texture analyzer (Lloyd Instruments, Ltd., West Sussex, UK). The samples were cut into small cubes of 2 × 2 cm on both sides. TPA was determined according to the program: pretest speed: 0.5 mm/s; test speed: 5 mm/s; and trigger force: 0.05 N.
The fish sample was subjected to two cycle's compression up to 30% of its original height using a 12-mm diameter cylindrical probe. The measurement was performed in triplicate.

| Total volatile basic nitrogen (TVB-N)
The TVB-N was measured after perchloric acid distillation from homogenized fish fillet samples (Abelti, 2013). The distillate was recovered in an Erlenmeyer flask containing aqueous solution of 20 g/L boric acid and some drops of methyl red as an indicator. Then, the boric acid solution was titrated with 0.1 M sulfuric acid solution. The TVB-N (mg N/100 g sample) was measured based on the volume of sulfuric acid used for titration according to the following where V is the volume of sulfuric acid consumed for titration, N is the normality of the sulfuric acid, and m is the sample mass.

| Lipid peroxidation
The thiobarbituric acid reactive substances (TBARS) of fish samples were measured as described previously by Witte et al. (1970). Briefly, 5 g of fish sample was homogenized in 20 ml of 5% trichloroacetic acid solution using a Polytron PT 2100 homogenizer (Kinematica AG, Luzern, Switzerland) for 5 min. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant (4 ml) was reacted with 0.8 ml 0.6 M chlorhydric acid and 3.2 ml Tris-thiobarbituric acid (TBA) solution (26 mM Tris, 120 mM TBA) and then incubated in a water bath at 85°C for 10 min. The absorbance of each mixture was measured at 532 nm. TBARS values were calculated from a standard curve of malondialdehyde (MDA) and expressed as mg MDA/kg fish sample.
2.6.7 | Microbiological analysis Bacteriological counts were measured by mixing 1 g of fish sample in 9 ml of 0.9% NaCl solution, then appropriate decimal dilutions were prepared. The mesophilic and psychrotrophic counts were measured using plate count agar medium. The inoculated plates were incubated at 37°C for 2 days for the mesophilic bacteria and at 4°C for 7 days for the psychrotrophic bacteria. Iron and de Man, Rogosa, and Sharpe agar were used to enumerate H 2 S-producing bacteria (incubation at 37°C for 48 h) and lactic acid bacteria (LAB) (incubation at 30°C for 72 h). All bacterial counts were converted to logarithms of colonyforming units per gram of fish fillet (log 10 CFU/g) (Nowzari et al., 2013).

| Sensory analysis
Sensory evaluation of fresh fish samples was performed by 30 panelists who give a score for color (10 = no discoloration; 1 = extreme discoloration), odor (10 = extremely like; 1 = extremely unacceptable/off-odors), and overall acceptability (10 = extremely like; 1 = extremely unacceptable). For each analysis day, fillet piece for each treatment was placed in the individual booths, which had a random three-digit blind code and presented in the unsystematic order (Naeeji et al., 2020).

| Statistical analysis
One-way analysis of variance (ANOVA) was done using the statistical package for the social sciences (SPSS) software for Windows™ (version 17, SPSS Inc., Chicago, IL, USA). Duncan's multiple range test was used to compare the measured responses for different fish samples. Differences between means at the 95% (p ≤ .05) confidence level were considered statistically significant.

| Bioactive compounds profile
The bioactive compounds of M. oleifera leaves were extracted using three solvents: ethanol, ethanol/water (50/50, v/v), and water. Total phenolics and flavonoids contents were presented in Table 1. The ethanol/water extract showed the highest content of total phenolics (90.13 mg GAE/g extract) and flavonoids (16.77 mg QE/g extract) as compared to the ethanol and water extracts. Prabakaran et al. (2018) reported comparable levels of total phenolics (90-112 mg GAE/g extract) and flavonoids (55-69 mg QE/g extract) in alcoholic and aqueous extracts of M. oleifera leaves.
The LC-ESI-MS analysis of M. oleifera extracts was also assessed and profiles of phenolic compounds are shown in Table 1. Fourteen compounds distributed into seven phenolic acids (compounds 1-6 and 10) and seven flavonoids were identified by comparing the obtained mass spectra with those of 32 authentic standards of phenolic compounds. However, if the analyzed extracts contained different compounds from the used standards, they cannot be identified. have studied the profiles of phenolic compounds from the same leaf sample, but dried at 50°C (Mezhoudi et al., 2022). Similar profiles were obtained, but with lower contents for quinic acid (13.5 mg/g extract) and gallic acid (6.8 mg/g extract). In fact, the increase in temperature during oven drying (50°C) showed a decrease in quinic and gallic acids by 57% and 81%, respectively, which could be attributed to the heat sensitivity of these compounds.  (Wink, 2003).
Besides, Nobossé et al. (2018) reported that the contents of bioactive compounds and antioxidant activity of M. oleifera leaves were influenced by the plant age and the extraction solvent used. It was reported that mixture of alcohol/water or acetone/water are the best extraction solvents with respect to the extraction yields of polar phenolic acids (Stalikas, 2007).
Quinic acid is a cyclic polyalcohol representing an important biochemical intermediate of the shikimate pathway, involved in the biosynthesis of aromatic compounds in plants (Herrmann & Weaver, 1999). Consumption of quinic acid as a dietary supplement was reported to increase the synthesis of nicotinamide and tryptophan in the gastrointestinal tract, which inhibits nuclear factor kappa B (NF-κB) and improves DNA repair (Pero et al., 2009). In addition, quinic acid exhibits powerful antioxidant, hepatoprotective, anti-inflammatory properties, as well as other interesting medicinal properties (Pero et al., 2009;Xiang et al., 2001). In the same context, it has been suggested that quinic acid could be used as a potent drug candidate against prostate cancer (Inbathamizh & Padmini, 2013).

| Antioxidant potential
The abundance of phenolic compounds in M. oleifera leaves could confer an interesting antioxidant potential, since these compounds have the ability to stabilize the free radical generation by their reactivity as electron-or hydrogen-donating molecules. Thus, the extracts were subjected to their antioxidant activities, which were evaluated by reducing (Fe 3+ ) power and DPPH• radical-scavenging assays (  (Table 1) oleifera or food products supplemented with the leaves of this plant is likely to provide antioxidant potential and consequently health benefits as reported previously (Singh et al., 2020).

| Antibacterial properties
The antibacterial activity of M. oleifera extracts against eight species of microorganisms was evaluated by the inhibition zones determination ( Table 2). The different extracts presented varying degrees of antimicrobial activity against the tested strains. The highest inhibition zones for the most strains were obtained for the ethanol/ water and water extracts, which suggest that these extracts may be useful in inhibition of many microorganisms. Prabakaran et al. (2018) reported the antimicrobial activity of five solvent extracts from dif- The numbering refers to elution order of compounds from an Aquasil C18 column.
b Identification was confirmed using 32 authentic commercial standards. The presence of many phenolic compounds may explain the observed antimicrobial activity. In fact, Fu et al. (2016) reported that various phenolic compounds (gallic acid, caffeic acid, ferulic acid, quercetin, and luteolin), which were identified in the studied extracts, have interesting antimicrobial activity. The antibacterial activity of phenolic compounds should include many mechanisms, such as disruption of cell membrane structure and permeability and inhibition of enzymes necessary for nucleic acid synthesis leading to cell death (Gill & Holley, 2006;Gradišar et al., 2007). Besides, one can notice that synergistic and antagonistic effects between several phenolic compounds in the extract should also be taken into consideration.
Thus, phenolic compounds identified in M. oleifera leaves could be used as a potential natural antibacterial and antioxidant agent to control food poisoning diseases as well as oxidation. In the following, the ethanol/water extract was chosen as a potential extract to be added in the gelatin solution used to coat the smooth-hound shark fillets.  were used for structural modification of gelatin. Araghi et al. (2015) reported that caffeic acid was an effective phenolic compound that improves the barrier and physicochemical properties of gelatin packaging (Araghi et al., 2015).

| Physicochemical characteristics
The TVB-N developed in fish products during storage are widely used as chemical indicator of their spoilage, with a critical limit of 25-35 mg N/100 g fish fillet (Šimat et al., 2009). The initial TVB-N was determined to be 6.05-6.16 mg/100 g, which was close to the value (8.12 mg N/100 g) reported for fresh trout fillets (Eghbalian et al., 2021).   A similar effect of gelatin coating enriched with cinnamon (Andevari & Rezaei, 2011) and oregano (Hosseini et al., 2016) essential oils on reducing TVB-N content of stored rainbow trout fish. In the same context, sodium caseinate-gelatin nanofibers containing Mentha spicata essential oil decreased the TVB-N content of fresh trout fillets (Eghbalian et al., 2021). Coating with gelatin/phenolics was more effective than gelatin applied alone, which can be attributed to the microbial growth inhibitory activity exerted by the phenolic compounds in the M. oleifera extract.
Lipid peroxidation is also an important factor limiting the sensory properties of fish products. It was reported that 1-2 mg MDA/kg is usually regarded as the limit TBARS in fresh fish fillet (Connell, 1990). Thus, TBARS were measured during the storage period ( Figure 1). The initial TBARS value was 0.60 mg MDA/kg fish fillet, which was close to the value of fresh smooth-hound shark fillets reported in an earlier study (Abdelhedi et

| Texture and color characteristics
The texture properties of the fish fillets are important sensory parameters for consumers' acceptability. Table 3 shows the values of texture parameters (strength, cohesiveness, springiness, and chewiness) in smooth-hound shark stored during 6 days after different coating treatments. As in case of weight loss, the strength of all fish fillets significantly increased (p ≤ .05) during 6 days of storage, which might be due to the exudate loss ( Figure 1). Interestingly, this increase was attenuated with gelatin coating. The strength of uncoated fish increased by 46%, while the strength of coated-fish with gelatin and gelatin-MOE/W only increased by 14% and 12%, respectively, after 6 days of storage ( Table 3).
Values of cohesiveness, springiness, and chewiness did not show significant differences (p > .05) between the coated samples or through the chilled storage within the same sample. Only slight variations throughout the chilled storage were observed within the uncoated sample. The obtained results suggest that gelatin coating did not result in important modifications in the texture properties of fish fillet during chilled storage. This seems to be supported by Gallego et al. (2020), who reported that gelatin coating enriched with antioxidant tomato by-products did not modify the texture properties of pork meat, as compared to the uncoated sample.
The color is also a crucial factor in food quality control, since consumers rely on color to determine the freshness level of the product.  lets was between 2.01 and 2.54 log 10 CFU/g, respectively, which was much lower than the threshold of 7.0 log 10 CFU/g for the maximum allowable limit of fish (Swanson, 2011). An increase in mesophilic and psychrophilic bacterial count was measured from day 1 to day 6 for all fish samples. Coating with gelatin significantly reduced (p ≤ .05) the number of these bacteria during storage. Moreover, coating with gelatin-MOE/W resulted in a reduction of ~1 log10 CFU/g in the number of mesophilic bacteria. Likewise, the initial count of LAB in uncoated fish fillets was 1.26 log 10 CFU/g (day 1) and reached 1.68 log 10 CFU/g at day 6 of refrigerated storage (Figure 2c).   to its role as a barrier against oxygen diffusion and subsequently bacterial proliferation. Besides, the antimicrobial activity of MOE/W extract added in the film could enhance the antimicrobial property of edible gelatin coating, which could be an effective way to extend the storage period of fish fillet.

| Sensory analysis
The sensory results (odor, color, and general acceptability) of fresh fillets are shown in Figure 3a-c. Sensory properties of all samples decreased throughout the storage period for all samples. As compared to the control group, coated samples showed higher scores for all attributes. The gelatin-MOE/W coated fish fillet had the highest sensory scores in odor, color, and overall acceptability over the storage period. In this regard, it was reported that trout fillets coated with sodium caseinate nanofibers added with Me. spicata essential oil showed better sensory properties compared to the control sample (Eghbalian et al., 2021).

| CON CLUS ION
Edible coatings enriched with natural bioactive compounds are increasingly in demand in the food industry. This study showed that fish fillet coated with a combination of fish gelatin and M. oleifera extract contributed to delay its deterioration during chilled storage.

ACK N OWLED G M ENTS
We thank the Ministry of Higher Education and Scientific Research (Tunisia).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All authors confirm that the data supporting the findings of this study are available within the article.